1. Field of the Invention
The invention relates to the field of calorimetry, and in particular, to a system and method for reducing nanocalorimetry operating costs.
2. Related Art
Calorimetry is used to measure enthalpic changes, including enthalpic changes arising from reactions, phase changes, changes in molecular conformation, temperature variations, and other variations of interest that may occur for a particular specimen. By measuring enthalpic changes over a series of conditions, other thermodynamic variables may be deduced.
For example, measurements of enthalpy as a function of temperature reveal the heat capacity of a specimen, and titrations of reacting components can be used to deduce the binding constant and effective stoichiometry for a reaction. Calorimetry measurements are useful in a broad variety of applications, including, for example, pharmaceuticals (drug discovery, decomposition reactions, crystallization measurements), biology (cell metabolism, drug interactions, fermentation, photosynthesis), catalysts (biological, organic, or inorganic), electrochemical reactions (such as in batteries or fuel cells), and polymer synthesis and characterization, to name a few.
In general, calorimetry measurements can be useful in the discovery and development of new chemicals and materials of many types, as well as in the monitoring of chemical processes. Standard calorimeters require relatively large samples (typically about 0.5 ml to 10 liters) and usually measure one sample at a time. As such, these systems cannot be used to measure very small samples, as might be desired for precious or highly reactive materials. Furthermore, standard calorimeters cannot be used effectively to monitor a large number of reactions of small sample size in parallel, as is required in order to perform studies using combinatorial chemistry techniques.
In recent years, researchers and companies have turned to combinatorial methods and techniques for discovering and developing new compounds, materials, and chemistries. For example, pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. As another example, Symyx Technologies™ is applying combinatorial techniques to materials discovery in the life sciences, chemical, and electronics industries.
Consequently, there is a need for tools that can measure reactions and interactions of large numbers of very small samples in parallel, consistent with the needs of combinatorial discovery techniques. Preferably, users desire that these tools enable inexpensive measurements and minimize contamination and cross-contamination problems.
One of the most popular uses of combinatorial techniques to date has been in pharmaceutical research. Pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. A combinatorial library is a collection of chemical compounds that have been generated, by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” as reagents. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixing of chemical building blocks.
Once a library has been constructed, it must be screened to identify compounds, which possess some kind of biological or pharmacological activity. For example, screening can be done with a specific biological compound, often referred to as a target, that participates in a known biological pathway or is involved in some regulation function. The library compounds that are found to react with the targets are candidates for affecting the biological activity of the target, and hence a candidate for a therapeutic agent.
Through the years, the pharmaceutical industry has increasingly relied on high throughput screening (HTS) of libraries of chemical compounds to find drug candidates. HTS describes a method where many discrete compounds are tested in parallel so that large numbers of test compounds are screened for biological activity simultaneously or nearly simultaneously. Currently, the most widely established techniques utilize 96-well microtitre plates. In this format, 96 independent tests are performed simultaneously on a single 8 cm by 12 cm plastic plate that contains 96 reaction wells. These wells typically require assay volumes that range from 50 to 500 microliters. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers and plate readers are commercially available to fit the 96-well format to a wide range of homogeneous and heterogeneous assays. To achieve faster testing, the industry is evolving to plates that contain 384 and 1536 wells.
A variety of measurement approaches has been used to screen combinatorial libraries for lead compounds, one of which is the inhibitor assay. In the inhibitor assay, a marker ligand, often the natural ligand in a biological pathway, is identified that will bind well with the target protein molecule. The assay requires the chemical attachment of a fluorescent molecule to this marker ligand such that the fluorescent molecule does not affect the manner in which the marker ligand reacts with the target protein. To operate an inhibitor assay, the target protein is exposed to the test ligands in microtitre wells. After a time necessary for reaction of the test ligand to the target protein, the marker ligand is applied. After a time for reaction with the marker ligand, the wells are rinsed such that non-reacted marker ligand is washed away. In wells where the target protein and the test ligand have reacted, the test ligand blocks the active site of the target protein so the marker ligand cannot react and is washed away, while in cells where the target protein and test ligand have not reacted, the marker ligand reacts with the target protein and is not washed away. By investigating the wells for the presence of fluorescence after the washing, reactions of test ligands and target proteins can be determined as having occurred in wells where no fluorescence is observable.
However, the inhibitor assay requires time and expense to develop the assay. The principal components that need development are discovering a marker ligand and attaching a fluorophore to the marker in a manner that does not affect its reaction with the target protein. Attaching the fluorescent marker can often take 3 months of development or more and cost $250 k or more once the marker ligand is identified. An assay method that avoids such assay development, such as measuring the heat of the reaction with calorimetry, would eliminate this cost and lime delay in the discovery process.
Calorimetry measurements are commonly utilized in biophysical and biochemical studies to determine energy changes as indications of biochemical reactions in a media. Prior techniques for measurements include using electrodes, thermopiles, optical techniques, and microcalorimeters for measurements within a sampled media. There is a great interest in developing calorimetry devices, and in particular, ultra-miniature microcalorimeter devices (i.e., nanocalorimetry devices), that require very small volumes of sampled media and that can quickly measure large numbers of reactions. Ideally, those reaction measurements can provide efficient assays; e.g., inhibitor assays which can be used in HTS to screen roughly 100,000 test ligands a day.
Accordingly modern calorimetry tools (in particular, microcalorimetry and nanocalorimetry tools) include an array of detectors that allow multiple measurement operations to be performed simultaneously. FIG. 1A shows a top view of a nanocalorimeter array 100, which is similar to nanocalorimeter arrays described in detail in co-owned, co-pending U.S. Patent Application Serial No. 2003/0186453, herein incorporated by reference.
Nanocalorimeter array 100 includes a frame 110 and two detectors 120. Detectors 120 are commonly amorphous silicon (a-Si) structures that are formed on a Kapton™ plastic film (shown in FIG. 1B), which in turn is supported by frame 110. Detectors 120 include the devices necessary to perform calorimetry measurements on sample droplets 190 of test material. The measurement data is then read out from detectors 120 via contacts 111 on the periphery of frame 110.
In addition, microcalorimeter and nanocalorimeter arrays, such as nanocalorimeter array 100, typically include a thin (1-3 μm) parylene coating 130 that is deposited over detectors 120 (and frame 110). Sample droplets 190 are placed directly onto parylene coating 130, which provides a hydrophobic surface that facilitates the merging and mixing of sample droplets 190. Parylene coating 130 also provides electrical and chemical passivation for detectors 120, while still allowing the thermal effects of droplet interactions to be measured by detectors 120.
The coverage provided by parylene coating 130 is more clearly depicted in FIG. 1B, which shows a cross section of nanocalorimeter array 100. As described above with respect to FIG. 1A, parylene coating 130 covers detectors 130, which in turn are formed on Kapton™ layer 131 (copper strips 132 beneath detectors 130 are isothermal elements to ensure that the measurement elements of detectors 120 are thermally coupled to sample droplets 190).
To avoid contamination, nanocalorimeter arrays (such as nanocalorimeter array 100) are typically discarded after a single use, which can add significant costs to large nanocalorimetry experiments. While parylene coating 130 can sometimes be cleaned and sterilized to enable re-use of nanocalorimeter array 100, such refurbishment activity can be time-consuming and expensive.
Accordingly, it is desirable to provide a system and method for performing calorimetry operations that enables reuse of calorimeter arrays.